Abstract. Previous studies from our laboratory have obtained evidence implicating sumoylation in regulation of different events during spermatognenesis. SUMO is abundantly expressed in germ cells from different species, and the localization pattern suggests diverse and potentially multiple roles of sumoylation in testicular function. We have also performed a broad-spectrum cell-specific identification of SUMO targets in purified mouse germ cells. Numerous proteins with important roles during mitosis, meiosis and spermatid differentiation have been identified. Level of sumoylation correlated with the proliferation activity of germ cells. Notably, in mouse spermatocytes, inhibition of global sumoylation with the inhibitor Ginkgolic acid (GA) arrested cells at the G2/M1 checkpoint in vitro due to the inability of spermatocytes to condense chromatin and disassemble their synaptonemal complexes (SC). Our preliminary data show that Polo kinase1 (PLK1) and Aurora B (AURKB) are activated upon G2/M meiotic transition, but significantly inhibited upon inhibition of sumoylation. Since PLK1and AURKB are responsible for the disassembly of the SC and chromatin condensation, respectively, these kinases are at the right place, at the right time to at least, in part, explain the meiotic arrest. However, definite evidence regarding the importance of sumoylation and its targets in germ cells in vivo is lacking because of the embryonic lethality of mice with inactivated sumoylation machinery. The specific aims of the current application will be: 1) To perform a cell- specific inactivation of sumoylation and characterize the consequences of this inactivation in mitotic spermatogonia, meiotic spermatocytes and differentiating spermatids in vivo using a UBA2 (SUMO- activating enzyme) loxP mouse model that we possess as well as commercially available mouse models expressing Cre recombinase in the corresponding germ cells; 2) To dissect the molecular events regulated by sumoylation in spermatocytes through analysis of the activity, interacting partners and phosphorylation status of known downstream targets of PLK1 and AURKB upon inhibition of sumoylation in vivo and in vitro. Together, the proposed research and follow-up studies will advance knowledge across the field of germ cell biology by the characterization of post-translational modification networks necessary for the progression throughout spermatogenesis. A significant part of the proposed project will be conducted by undergraduate students who will have the opportunity to use state-of-the-art equipment, learn hypothesis generation and practice basic and advanced biomedical techniques. They will be involved in germ cell isolation, preparation of cell lysates, western blot, PCR, microscopy, and bioinformatics analyses. Opportunities to participate in research early in their careers will give students a better understanding of a career in science and will motivate them to pursue their education in STEM fields.